Abnormal outer hair cell efferent innervation in Hoxb1-dependent sensorineural hearing loss

Autosomal recessive mutation of HOXB1 and Hoxb1 causes sensorineural hearing loss in patients and mice, respectively, characterized by the presence of higher auditory thresholds; however, the origin of the defects along the auditory pathway is still unknown. In this study, we assessed whether the abnormal auditory threshold and malformation of the sensory auditory cells, the outer hair cells, described in Hoxb1null mutants depend on the absence of efferent motor innervation, or alternatively, is due to altered sensory auditory components. By using a whole series of conditional mutant mice, which inactivate Hoxb1 in either rhombomere 4-derived sensory cochlear neurons or efferent motor neurons, we found that the hearing phenotype is mainly reproduced when efferent motor neurons are specifically affected. Our data strongly suggest that the interactions between olivocochlear motor neurons and outer hair cells during a critical postnatal period are crucial for both hair cell survival and the establishment of the cochlear amplification of sound.

specifica2on of IEE the strength of our mouse models, i.e., tes2ng the role of IEE neurons, altered by gene2c means, in the amplifica2on process of sound (differently than by lesions and/or axonal transec2ons).
Moreover, the purpose of our study is not to follow the fate or rewiring of Hoxb1-deficient ventral r4 neurons, but to understand whether the failed interacFon between MOCs and OHCs can explain the auditory threshold phenotype we describe in some mutant mice.We thus believe that the most reliable experiment to visualize OHC and MOC interacFons is electron microscopy (EM) and, parFcularly, Transmission EM (TEM), as shown in Figs. 3 and 7. TEM was performed exactly on the same mice tested for ABR, thus strongly correlaFng anatomical to funcFonal defects.The TEM analysis showed that in the CKONkx2.2 and CKOr4 mice with high thresholds, no or very few MOC/OHC interacFons could be detected in all secFons examined.The quanFficaFon has been added in the new Fig.S1 and S1 Data.
Regarding the tracing, we had already performed some retrograde tracing on P8 Hoxb1null and CKOr4 (previously called Hoxb1 lateCKO ) brains in our previous paper (Di Bonito et al., 2013; PMID: 23408898see Fig. 6).Both genotypes have no MOC neurons crossing the midline and no obvious redirecFon to the facial MNs, which are also highly affected.We think that dye tracing experiments become very tricky and variable in cases of just none or very few retrogradely-labeled axons.Since the IEE-derived phenotype of the CKOr4 looks very similar to the CKONkx2.2(see Fig. 4 in our present study), we do not expect any or very few axons to cross the midline.Again, we think that the TEM analysis, which confirms the presence or absence of OHC-MOC interacFons is more adapted for our purposes.
Reviewer: P11 line 16 why is a _ between cochlea and MOC? Authors: Thank you for poinFng out the typo.
Reviewer: References: 2 should be replaced by Malmierca, 2015 3 Simmons should be replaced by Simmons et al., 2011 Authors: We have added these two citaFons but also lee the Simmons 2002 one since this review nicely describes the development of IEE across vertebrate species.
Reviewer: 15 I believe the paper by Zuo et al., 1999  Authors: Thank you for the quesFon.There are no significant differences regarding threshold between wild-type and heterozygous mice, even for the 32 kHz of sound-sFmulaFon frequency (@ 32 kHz, Pvalue = 0.108742, two tailed Mann-Whitney-Wilcoxon test).In the new version of Fig. 1, the difference between genotypes regarding the thresholds and DPOAEs are indicated with stars (*<0.05,***<0.001and n.s.>0.05).
Reviewer: Fig. 2 A recent publica4on (EllioW et al., 2023) shows the expansion of Pl1a dele4on.It would be interes4ng to show the effect of Pl1 dele4on in r4 that should show less expansion compared to Pl1a dele4ons.
Authors: This is a very interesFng point, but we are just using the Pk1-Cre line to delete Hoxb1 in r4.To find out what happens if Pk1 is deleted in r4, we would need to generate a completely new mouse line, i.e.Pk1 floxed with b1r4-Cre, and a specific new study.
Reviewer: Fig. 4 A detailed tracing informa4on was presented by Bruce et al., 1997 that clarifies the presenta4on of ves4bular, LOC and MOCs.How many ves4bular efferents develop in different CKO dele4ons?Authors: We know from our previous paper (Di Bonito et al., 2015 eNeuro -Fig. 5) that vesFbular efferents are completely lost in the Hoxb1null mutant, as demonstrated by marker expression and retrograde tracing.We have not analyzed what happens with these neurons in the other mutants, but we would expect very few of these neurons in the CKOr4 and CKONkx2.2, and no difference in the sensory-deleted mutants, since we do not inacFvate Hoxb1 in IEE neurons.We do not consider that addiFonal analysis on vesFbular efferent in all mutants would reinforce our data on the interacFon between MOCs and OHCs, which is the main message of our study.Reviewer: Fig. 6 This informa4on seems to compare with Maricich et al., 2009 aDer Hoxb1 dele4ons of Atoh1.Different ABRs have been described aDer complete dele4on of Isl1 (Filova et al., 2023).How close is the ABR aDer different dele4ons?Authors: The ABR from the different deleFons (Hoxb1 null, CKOr4 and CKONkx2.2) did not differ between them regarding the amplitude and latencies of the ABR wave 1, 3 and 4. Comparing the mean waveforms of the ABR can be more difficult as the number of mice recorded, and the latency of the waves directly impact the average waveform.In addiFon, a comparison of the ABR waveforms from different labs can be tricky as the recording condiFons may substanFally differ (anesthesia, filters, transducers, rate of sound presentaFon…).We agree with the reviewer it would be interesFng to know the difference that can be hidden in the ABR from the different deleFons, but we would prefer not to overstate any conclusion.
Reviewer: Fig. 7. Nice images but will not proof the absence of MOCs.Suggest using a dye tracing to show the absence of MOCs (see Zuo et al., 1999).Authors: As menFoned above, we believe that TEM is an adapted tool for idenFfying the innervaFon of MOC neurons to OHC, parFcularly if there are only a few neurons innervaFng the hair cells.We think that dye added to the cochlea would label all fibers reaching the cochlea and that the synapses between MOCs and OHCs would be difficult to detect.Moreover, and importantly, our TEM analyses were performed exactly on the same animals which went through the ABR (with high and low thresholds), supporFng a strong correlaFon between the absence of MOC/OHCs interacFons and hearing abnormaliFes.
Reviewer: Fig. 8 Suggest providing Prph type II fibers (see Elliot et al., 2021).Show the immunostaining for type II afferents and trace the LOC/MOC differen4ally at P8 and P40.Authors: We thank the reviewer for this interesFng suggesFon, but we believe that this is outside the focus of our study, which is mainly to prove that MOC efferent fibers are the responsible cells for the abnormal auditory threshold observed in Hoxb1 mutants.Since Hoxb1 is not expressed in afferent fibers, we do not expect any changes in type II afferents.

Reviewer #2:
Reviewer: The manuscript is well wriWen, figures are clear, sta4s4cs are appropriate.Some more recent literature is missing from cita4ons (see detail below).Overall this is important work that furthers our knowledge of the role of the olivocochlear efferent system in maintaining cochlear cell health and hearing func4on by demonstra4ng that loss of olivocochlear neurons in developmental stages causes OHC degenera4on.Authors: We thank the reviewer for acknowledging the quality and interest of our manuscript.
Reviewer: Weaknesses of the work are that it depends on precise knockout of Hoxb1 in different cell popula4ons using different Cre driver lines.This work does not demonstrate the precision of the knockouts, readers must reference previous works to see demonstra4ons of the specificity of the mouse lines.However, a helpful schema4c is added to show how the cell-type specific knockouts are performed by targe4ng different rhombomeres in the developing nervous system.There are some variable results with the penetrance of the Cre in some of the condi4onal knockouts, resul4ng in some variable auditory func4on responses, but this is sufficiently discussed and analyzed in the manuscript.Authors: We agree with the reviewer that the schemaFc helps in understanding the type of deleFons we have produced with the different mouse lines.
Reviewer: Another weakness is that there is not enough evidence to demonstrate that MOC efferent terminals are consistently aberrant in the cKO animals, more detail below.Finally, the authors do not convincingly demonstrate that the effects of knockouts are limited to MOC, not LOC neurons or with contribu4ng effects from the middle ear muscles.Contribu4ons from these other systems may indeed contribute to pathology and are also an important finding, but the language regarding the contribu4ons of these other systems to pathology should be more clear.Authors: We fully agree with the reviewer and have clarified in the discussion the reasons why we think that LOC neurons are most probably unlikely to parFcipate in the increase of threshold levels we observed in Hoxb1 mutant mice.However, we acknowledge that LOC neurons might be involved in regulaFng the amplitude of the waves (pages 12-13), as suggested by lesion experiments (Darrow et al., 2006 -PMID: 17115038 and2007 PMID: 17093118).Moreover, we cannot exclude that facial MNs might also contribute to the auditory phenotype we have described in the affected mutants.We hypothesize that the absence of the efferent reflex system (MEM and MOC) renders Hoxb1 mutant cochleae more hypersensiFve to environmental sounds and that the OHCs (and consequently the cochlear amplificaFon system) might result increasingly damaged with Fme determining a more pronounced progressive age-related degeneraFon of hearing in Hoxb1 mutant mice, as previously described (Di Bonito et al., 2013 PMID: 23408898).
Reviewer: Major: • Evidence of MOC axon terminal degenera4on: The transmission EM images showing degenera4on of MOC efferent terminals onto OHCs in the null and 'high' lines requires at a minimum more detail of how many sec4ons were analyzed because it is possible that the examples shown are simply from a sec4oning plane below OHCs that does not happen to have an MOC terminal.While this is unlikely, more descrip4on of the process of examining mul4ple sec4ons through OHCs is necessary to thoroughly eliminate the possibility that MOC neurons are healthy but in other sec4ons, or MOC neurons are unhealthy and degenera4ng, or small, but present, which would add addi4onal detail to the research.At present, only a single animal is used for each condi4on, with only a single example image shown and liWle detail regarding numbers, which is simply not enough evidence.More detail is required about the experiments that were performed.Authors: We fully appreciate the reviewer's concern and apologize for not having clarified that the analysis was performed on mulFple secFons of mulFple animals and not only on one animal per genotype, and that all the secFons including OHCs have been carefully analyzed for the presence/absence of MOCs.We have now quanFfied the percentage of MOC/OHC interacFons on Ctrls (n=3), Null (n=3), CKONkx2.2 high and low (n=6) and CKOr4 low and high (n=6).The new staFsFcs have been added in the text and in Fig. S1 (see also S1 Data) and the numbers of samples added on page 17.
Reviewer: I would also recommend whole mount immunolabels with OC markers such as VAChT or CHAT or AChE to show a loss of MOC terminals across many more OHCs.Authors: We fully appreciate the reviewer's suggesFon, but we are not sure that these markers would label the MOC/OHC interacFons as precisely as the TEM analysis.Moreover, we deeply apologize but are unable to perform these experiments because all our mouse lines had to be cryopreserved due to the refurbishment of our animal facility.
Reviewer: Lack of effects of cKO on LOC efferents, middle ear muscles, central neurons.In these experiments LOC efferent neurons will also be disrupted at the same 4me as MOC neurons.The loss of LOC neurons may also contribute to the hearing deficits observed here.Is there a way to more clearly determine whether the LOC neurons are involved, or not?Authors: We fully agree with the reviewer that in our experiments MOC and LOC neurons are both affected.To our knowledge, there is no way to find out whether LOC neurons alone are responsible for the Hoxb1 hearing phenotype since we would need a Cre-recombinase mouse that specifically inacFvates LOC neurons.Unfortunately, we do not know the existence of such a mouse.As menFoned above, we have now beqer clarified in the discussion (pages 12-13) why we believe that LOC neurons are unlikely to contribute to the increased threshold levels found in mutant mice.However, we cannot exclude (and actually we propose it) that absence of LOC neurons might be instead involved in amplitude changes (see Fig. 6), as previously suggested by the group of Prof. Liberman (Darrow et al., 2006 -PMID: 17115038 and2007 PMID: 17093118).
Reviewer: Similar to the sugges4on above, cholinergic staining in the inner spiral bundle may assess LOC innerva4on.Authors: We fully appreciate the reviewer's suggesFon, but as menFoned above we do not have live animals anymore.
Reviewer: Also, more detailed analysis of ABR wave 1 amplitudes, latencies, etc may be used to assess changes to the func4on of the type I SGN independent of thresholds and OHC func4on.Authors: We agree with the reviewer that a detailed analysis of ABR amplitude and latency independently of the threshold and OHC funcFon is meaningful, notably to study the inner hair cell and/or the SGN funcFoning.However, to assess auditory funcFon, we usually try to avoid repeFFve high-level sound sFmulaFon to minimize any trauma that could be elicited during the recordings.Thus, we did not probe the input-output funcFon at a high level of sFmulaFon (95 to 100 dB SPL), in which the acFvity within the cochlea is predominantly driven by the moFon of the basilar membrane (a passive mechanism) and not by the OHCs.
Reviewer: Some of the wording in the manuscript includes the possibility that LOC efferents play a role in auditory dysfunc4on (eg second to last sentence in the introduc4on) while other lines exclude LOC func4on (eg the final line of the introduc4on), and these should be clarified throughout.Authors: We apologize for the confusion; we have now uniformed our statements in the IntroducFon and Discussion.Reviewer: The figure 1 colors look ok in a pdf, but they print poorly -consider using a different color for the Hoxb1null animals, or at least a different shade of blue.In addi4on, using different symbols in plots B and D for the different genotypes would aid readability.Authors: We agree with the reviewer.Figures have been improved according to the suggesFon of the reviewer.
Reviewer: The text throughout figure 2 is too small Authors: We have now enlarged the genotypes and some of the text.Hope this is acceptable now.
Reviewer: Error bars appear to be either missing in plots 2 F and G, or are too small.I would expect them to be far larger than in plots C-E because there is obviously more spread in the data.Authors: We agree with the reviewer, sorry for that.In plots 2 F and G, error bars were small and hidden behind marker symbols.We corrected the plots 2C-H with smaller marker symbols.Error bars are visible now.

Reviewer #3:
The study is important because HOXB1 muta4ons in humans cause hereditary hearing impairments.Hoxb1 global muta4ons in mice also cause hearing loss, as earlier work by the authors has showed.The current study extends their previous work by using condi4onal mouse mutagenesis to pinpoint the cellular basis of Hoxb-1-linked hearing loss.The data strongly suggest that the interac4ons between olivocochlear motor neurons and outer hair cells during a cri4cal postnatal period are crucial for the establishment of the cochlear amplifica4on of sound.Major strengths of this work include: (a) four condi4onal mouse mutants are characterized, (b) Thorough electrophysiological analysis and high-resolu4on anatomical methods are employed, (c) Side-by-side comparison of condi4onal and null Hoxb1 mutant mice, and (d) the wide distribu4on of audiogram is nicely analyzed with hierarchical classifica4on in all mouse mutant lines.Authors: We thank the reviewer for highlighFng the strength of our study.
Reviewer: A number of weaknesses we are also iden4fied, as described below.Most (but not all) conclusions are supported by the data.Major issues: 1. Title: Improve 4tle of the paper because the causal role of Hoxb1 in genera4ng the innerva4on phenotype is not clearly reflected.Authors: We have now changed the Ftle to "Abnormal outer hair cell efferent innervaFon in Hoxb1dependent sensorineural hearing loss".
Reviewer: The Introduc4on and Discussion can be condensed and improved.2. Introduc4on: The authors are encouraged to condense and simplify the first paragraph of Introduc4on.Please, provide only the necessary informa4on of cell types needed to understand the paper.Avoid the use of 10 acronyms (OHC, IHCs, VIII, CN, LL, LOC, MOC, IEE, MEM, PVCN).Are all these necessary to be men4oned for the reader to understand the paper?It seems that all the reader needs to know to understand the paper is MOC (motor neurons) and OHC (sensory neurons).Authors: We have now simplified the first paragraph and reduced the number of acronyms, as requested.Thank you for the suggesFon.It reads much beqer.
Reviewer: 3. Discussion: Discussion is unnecessary long and needs a lot of work to be improved.This is important.The authors are strongly encouraged to reduce it to 3-4 pages maximum (current length is 6 pages), so the key take-home messages are succinct and easy for the reader to grasp.The first 2 pages of the Discussion add new informa4on from the literature full of acronyms and anatomical terms.This part en4tled "Development and func4on of efferent innerva4on…"is not necessary, and I am not sure what the discussed nAChR studies add to this paper.These 2 pages can be subs4tuted with a short paragraph that tells the reader what is the knowledge gap, and what is the new informa4on that this manuscript provides.The authors are encouraged to follow this guide: hWps://journals.plos.org/ploscompbiol/ar4cle?id=10.1371/journal.pcbi.1003453Authors: We fully agree with the reviewer and apologize for the long discussion.We have now shortened it to 3 and a half pages and kept the key take-home messages.We have also clarified the different roles of the MOC and LOC neurons, and the main reasons for our conclusions on the key role of MOC neurons on the Hoxb1 auditory phenotype.Reviewer: 4. A key message of the paper is that interac4ons between motor neurons and OHCs are crucial.Pre-synap4c and post-synap4c marker analysis would significantly strengthen this conclusion.Also, in the text and Abstract, the authors should make clear what do they think is the nature of this interac4on.I presume it is a direct (chemical) synapse.Authors: We agree with the reviewer that a careful analysis of pre-and post-synapFc markers on whole cochleae would strengthen our conclusion, even if we strongly think that electron microscopy is a more adapted strategy for detecFng close interacFons between the two cell types.We are unfortunately unable to do the suggested experiments since we have no mice alive due to the refurbishment of our animal facility.
Reviewer: 5. Essen4al to the conclusions of the paper is the detailed characteriza4on of the condi4onal mouse mutants.The Nkx2.2 CKO line is par4ally characterized.The confirma4on of reduced Hoxb1 mRNA expression in Nkx2.2Cre mice in ventral r4 is convincing, but the authors conclude, based on Tbx20, that a decreased popula4on of LOC and MOC neurons is present.However, it remains unclear whether LOC and MOC neurons are normally born but fail to differen4ate and extend axons and dendrites, or they are not born at all, or born and then die.Experiments with addi4onal MN markers (GATA3, ChAT) or cell death markers could help dis4nguish between these possibili4es.Authors: Our earlier experiments on Hoxb1null and Hoxb1CKOr4 (see Fig. S5 in Di Bonito et al., 2013, PMID: 23408898) have shown specific loss of Gata3 (early marker of IEE) and Chat on E10.5 and E14. 5 mutant embryos.Below the reviewer can find an addiFonal figure on coronal secFons through r4 illustraFng the loss of ventral Gata3 in differenFaFng motor neurons (Phox2b+/Islet1+) and no parFcular increase of death cells in mutant embryos.At these early stages, it is very difficult to disFnguish a few OC cells in condiFonal embryos.Since Hoxb1 is mainly implicated in impinging an r4 idenFty in the hindbrain (reviewed in Di Bonito et al. 2013, PMID: 23996673 and2017 PMID: 28469562), and IEE (including MOC and LOC) are exclusively generated in ventral r4, the absence of Hoxb1 in ventral r4 will convert the idenFty of r4-specific motor neurons into a ventral r3-like idenFty, which will result in the loss of IEE neurons since r3 does not generate IEE neurons.Finally, since Nkx2.2 is expressed in r4 progenitors from E10.5 (see Paqyn et al., 2003, PMID: 12651891), we are quite confident that the absence of Hoxb1 exclusively in ventral r4 upon Nkx2.2-Cre acFvaFon would result in the same phenotype as Hoxb1CKOr4, as also confirmed by the absence of Hoxb1 itself in ventral r4 (Fig. 4A in this study).
Reviewer Fig. 1: Coronal secFons through r4 at E10.5 in control (Ctrl) and Hoxb1 mutants (Hoxb1CKO and Hoxb1null).Note the strong reducFon of motoneuron progenitors and differenFaFng cells (arrows in Phox2b/Islet1), the loss (asterisks) of IEE precursors (Gata3), and no parFcular increase of apoptoFc cells (Caspase3).Since Gata3 staining does not label only the OC, but also other cells in adjacent zones (see Fig. S5 in Di Bonito et al., 2013, PMID: 23408898), it becomes difficult to disFnguish the few residual OC neurons in condiFonal mice at early stages.For this reason, we preferred to use Tbx20 at late stages (P8) in this paper.While Tbx20 staining in Ctrl brains in Figure 4 nicely disFnguishes LOC and MOC neurons in relaFon to their ventral locaFons, only a few cells Tbx20-expressing cells are present in the correspondent region in Hoxb1 condiFonal mice but not in Hoxb1null.However, Hoxb1 CKO mutants show also ectopic dorsal Tbx20 expression (see Fig. 4 in the manuscript and also Fig. 2 below), that probably are r3-like misspecified motor neurons, in agreement with the r4 to r3-like change of idenFty and with the evidence that Tbx20 is also expressed the developing motor trigeminal neurons that normally derive from r3 (see also Song et al., Development, 2006, PMID: 17119020).This part has now been clarified on page 8.
We would like to clarify that this change of ventral r4 iden7ty which prevents specifica7on of IEE highlights the strength of our mouse models, i.e., tes7ng the role of IEE neurons, altered by gene7c means, in the amplifica7on process of sound (differently than by lesions and/or axonal transec7ons).
Reviewer Fig. 2: In situ hybridizaFon of coronal secFons of Ctrl and Nkx2.2CKOP8 brains showing Tbx20 staining in medial (MOC) and lateral (LOC) olivocochlear neurons as well as in the trigeminal nucleus (V).Note that in the Nkx2.2CKO,Tbx20+ cells are sparser and posiFoned in different ectopic locaFons (arrows in the boqom right image) than in control (Ctrl) brains.We do not know the exact idenFty of these cells, but in light of the r4 to r3like change of idenFty upon Hoxb1 absence, we think that they might be r3-like misspecified motor neurons since Tbx20 is also expressed in the developing V nucleus that normally derives from r3.
Reviewer: Moreover, in Figure 4, quan4fica4ons are needed of the number of Tbx20-expressing LOC and MOC neurons, similar to what the authors did in their previous 2013 work (PMID: 23408898).The issue of what exactly happens to LOC and MOC neurons is par4cularly important in light of the observa4on that audiograms in CKONkx2.2 line are split 50/50 in high-and low-threshold.Authors: The P8 Tbx20 mRNA staining in Fig. 4 can vary from one embryo to the other and it becomes very difficult to quanFfy sparse Tbx20+ cells since they do not form a compact nucleus.These data on Tbx20 confirm earlier results in Hoxb1null and CKOr4 embryos described in our 2013 paper (Fig. S7) that were also not quanFfied.At this stage, it is very difficult to understand which pup will have a normal or abnormal auditory threshold.In all CKONkx2.2 pups examined we do see few or very few Tbx20+ cells with a variable paqern probably due to the Fming of acFon or parFal acFvity of the Crerecombinase (see also figure above).We think that it is this Fming or acFvity difference between Cre+ embryos that will produce high-or low-threshold groups, as we menFoned on page 9.Moreover, we have added in the new Fig.S1 a precise quanFficaFon of MOC/OHC interacFons detected by TEM.We believe that this is very relevant since the mice used for electronic microscopy are the same ones tested for ABR, allowing a strict correlaFon between the absence or presence of MOC and hearing phenotype.Finally, by revising all the raw data that we have now added in the S1 data Excel file, we noFced some small mistakes in grouping the different genotypes.Our new analysis now confirms an even stronger phenotype among the different mouse lines, parFcularly in the CKONkx2.2 group in which 67% of these mice fall into the high-threshold category while only 33% belong to the low-threshold class (differently from the 50-50 reported before) (revision in Fig. 5 and on page 9).Reviewer: 6.The manuscript men4ons that OHCs degenerate (do not survive) in the the Nkx2.2CKO line.Convincing data showing degenera4on of these cells are not provided.Authors: The reviewer is absolutely correct.We have now changed "degeneraFon" to "malformaFon" along the text, which is what we see in the SEM pictures.Reviewer: 7. Data are not provided to show loss of Hoxb1 expression in sensory (inhibitory and excitatory) cochlear neurons in CKOAtoh1 and CKOPl1a mice.This is very important in light of the absence of a phenotype in these animals (Fig. 2C-E).Authors: We fully understand the reviewer's concern.We have performed whole-mount ISH analysis also on early CKOAtoh1 and CKOPk1a embryos (at the same stage as the ones shown in Fig. 4), but even aeer the secFoning loss of Hoxb1 expression on these embryos was not very obvious.We think this is the case because the sensory populaFons posiFve for Atoh1 and Pk1a in r4 are very specific and most probably generated at a later Fme (than ventral motor neurons) and the whole-mount technique is not sensiFve enough to detect them.Since the acFvity of the Atoh1Cre and Pk1aCre lines on cells of the cochlear nucleus has been very well characterized in the original paper (Fujiyama et al 2009), as well as the reliability of Hoxb1floxflox mice, as demonstrated in our previous paper (Di Bonito et al 2013), we do not envisage any reason why these lines should not work.
Reviewer: 8. Quan4fica4ons of data (as they authors did in PMID: 23408898) presented in Figure 3 and Figure 7 would strengthen the conclusions.Authors: We fully agree with the reviewer and have now added the quanFficaFon of the percentage of MOC/OHC interacFons in Fig. S1 and individual data are listed in S1 data.
Reviewer: Minor issues: In Figure 1, panel A, The authors should bring the schema4c (WT only) from Figure 8.This would help the non-expert reader with auditory anatomy.It would also help communicate the key take-home message of this paper, i.e., the interac4on between motor neurons (MOC) and OHCs.Authors: We have now added a schemaFc in Figure 1 to help the non-expert reader to follow the different auditory steps.Figure 8 has been also updated.
Reviewer: In Introduc4on, last paragraph, please clarify that null phenotype is par4ally (not completely) reproduced in CKOr4 and CKONkx2.2 mutants.At the end of Results sec4on, it is also important to clarify this as well.Authors: We have now added "parFally" to beqer clarify the phenotypes observed in the CKOr4 and CKONkx2.2 mutant mice.
Reviewer: Minor: • Some references should be added: o Intro line 13, LOC effects -add Groff and Liberman 2003 PMID: 14615429.o Intro 15-16 MOC effects, add Romero and Trussell 2021 PMID: 34250904 for recent evidence of CN innerva4on of MOC neurons, also consider that MOC neurons receive inhibi4on: Torres Cadenas et al 2020 PMID: 31719165 o Intro lines 17-18 and discussion page 15 -consider recent work showing that the MEM reflex is more sensi4ve than previously appreciated, incorporate into work as necessary -2 works by Valero PMID: 26657094 and PMID: 29598837 -does this influence your interpreta4on of your results?o Intro first paragraph, last line -consider adding more recent works from the lab of Dr. Gomez Casa4 o Intro page 4 -"intermediate part of the dorsal cochlear nucleus" is not a regularly used term, what does this refer to?Granule cell domains separa4ng VCN and DCN? Granule cell domains within deep layers of the DCN? Other?o Discussion first paragraph, last line on page 11 -add Frank et al 2023 for neurotransmiWers in efferent neurons PMID: 36876911 OK o In mul4ple loca4ons that discuss the effects of the MOC efferent system on development of central neurons Clause et al should be referenced PMID: 24853941 Authors: We have added most of the references suggested by the reviewer.Thank you very much for the suggesFons.However, since we have simplified and shortened the introducFon and discussion parts, as requested by reviewer 3, some references were not necessary anymore.Reviewer: Typos: o Intro last line, 'func4ona4ng' to 'func4oning' o Intro page 4, first full paragraph, line 6 -'evolu4on' to 'evolu4onarily' o Intro page 5, 'previous to hearing func4onality' to ''prior to hearing func4onality' o Discussion first paragraph -an underscore is present instead of a space before 'MOC' in the middle of the paragraph.Authors: All these mistakes have now been corrected.We apologize to the reviewer.Reviewer: Figure 1 legend -'n indicates the number of cochleae recorded' should be included in the panel (D) text, not panel (E) Authors: The legend has been changed according to the comment and the revised Figure.Thank you for noFcing it.
should be cited here 19.suggest adding the cita4on of EllioW et al., 2021 28.suggest adding the work of Matei et al., 2005 for complete 30 a most recent presenta4on by EllioW et al., 2023 31 sugges4ng cita4ons such as Bruce et al., 1997 34 suggest adding the work of Karis et al., 2000 (2001) 55 suggest adding the cita4on of Wang et al., 2005 65 suggest adding for Wong et al., 2011 that details the feedback for Hox Authors: All these references have been added as requested by the reviewer.